1. Field of the Invention
The present invention relates to microscopes with higher resolution with partial spatial superposition in the illumination by an excitation beam and a de-excitation beam and/or a switching beam in a fluorescing sample.
2. Description of Related Art
Methods for increasing the optical resolution in a far-field with diffraction-limited resolution, which methods are based on nonlinear interaction of light with a sample, are known. Among such methods are microscopy with Stimulated Emission Depletion (STED) as discussed in U.S. Pat. No. 5,731,588, Ground State Depletion (GSD) as discussed in Hell and Kroug, Appl. Phys. B 60 (1995), pages 495-497, and optical modification (switching) of fluorescent substances as discussed in U.S. Pat. No. 7,064,824 B2.
In both cases, a diffraction-limited distribution of optical light (de-excitation light) is so modified by the diffraction-limited excitation distribution through a nonlinear interaction that the emission of the light can take place only from a sub-region. This sub-region (effectively Point Spread Function: PSF) can thereby be limited in all three spatial directions or only in a lateral direction. Suitable limits of the PSFs are thereby:                1. 3D limitation on the excitation volume; and        2. Lateral limitation of the excitation volume with enlarged depth of focus.        
Thus, the prior art, which is not so efficient from the viewpoint of the steepness of the gradient, involves the use of phase masks in the pupil of the de-excitation light with radial phase shift (See for example the article written by T. A. Klar, S. Jakobs, M. Dyba, A. Egner, and S. W. Hell, as published in Proceedings of National Academy of Sciences, U.S.A. 97, page 8206 (2000)) or of phase masks with quadrant-wise phase shift (In the article written by E. Engel et al. in Appl. Phys. B 77, pages 11-17 (2003)).
The use of the spiral masks in the STED microscopy was proposed in Török and P. R. T. Munro, Opt. Expr. 12 (2004), page 3605. However the use of a spiral mask, as described in the aforementioned article, generates neither an excitation distribution that is limited in all spatial directions, nor a distribution that exhibits enlarged depth of focus, as described in the following.